US20130209450A1 - Compositions and Methods for Treating Glioblastoma GBM - Google Patents

Compositions and Methods for Treating Glioblastoma GBM Download PDF

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US20130209450A1
US20130209450A1 US13/520,452 US201113520452A US2013209450A1 US 20130209450 A1 US20130209450 A1 US 20130209450A1 US 201113520452 A US201113520452 A US 201113520452A US 2013209450 A1 US2013209450 A1 US 2013209450A1
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Yael Cohen
Livnat Bangio
Andrew J. Brenner
Eyal Breitbart
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Definitions

  • the present invention in some embodiments thereof, relates to compositions and methods for treating malignant gliomas and, more particularly, but not exclusively, for treating Glioblastoma multiforme (GBM).
  • GBM Glioblastoma multiforme
  • Malignant gliomas the most common adult-onset neurological neoplasms, encompass a family of primary central nervous system tumors including glioblastoma, astrocytoma, oligodendroglioma, and ependymoma, along with the juvenile onset neoplasms such as juvenile pilocystic astrocytoma.
  • Malignant gliomas are typically characterized by over-expression of growth factors/tumor associated antigens believed to significantly contribute to the unchecked growth of such tumors.
  • Various malignant gliomas such as glioblastomas, exhibit epidermal growth factor receptor (EGFR) overexpression leading to increased aggressiveness and poor prognosis.
  • EGFR epidermal growth factor receptor
  • Malignant gliomas may also display over-expression of platelet-derived growth factor receptor, a phenomenon which has also been correlated with increased malignancy and poor prognosis.
  • Malignant gliomas the most common type of primary brain tumors, are aggressive, highly invasive, and neurologically destructive tumors which are among the deadliest of all human cancers. Of the estimated 17,000 new brain tumors diagnosed each year in the United States, about half are malignant gliomas. Malignant glioma cells produce very invasive brain tumors with infiltration of both white and gray matter. At the time of diagnosis, microscopic extension through much of the neural axis by malignant glioma is the rule. Such extension by motile invading cells underlies the incurability by surgery of most gliomas, even when they appear small and restricted in nature.
  • Glioblastoma multiforme the most serious form of malignant glioma, are extremely aggressive brain tumors which generally arise in the upper brain (cerebrum), but which may also occur elsewhere in the central nervous system, such as in the spinal cord, cerebellum, brain stem, or optic chiasm.
  • Low-grade gliomas which include astrocytomas, oligodendrogliomas, and pilocytic astrocytomas, account for 25% of all primary brain tumors, and over time most of these low-grade tumors dedifferentiate into more malignant gliomas.
  • Diffuse astrocytomas are predominantly located in the cerebral hemispheres of adults and have an inherent tendency to progress to anaplastic astrocytoma and (secondary) glioblastoma. The majority of glioblastomas develop de novo (primary glioblastomas), without an identifiable less-malignant precursor lesion.
  • Neovascularization is a major feature of glioblastomas (Maher et al., 2001, Genes Dev. 15:1311-1333).
  • Angiogenesis activators are extremely important in tumor growth, as reflected by the fact that neovascularization must occur for solid tumors to grow beyond a diameter of 2-3 mm (Goldbrunner et al., 2000, J. Neurooncol. 50:53-62).
  • One of the molecules that regulates this process is the vascular endothelial growth factor (VEGF).
  • VEGF mRNA is overexpressed in the highly vascularized glioblastoma multiform (Maher et al., 2001, Genes Dev. 15:1311-1333).
  • Bevacizumab (Avastin®) is a humanized monoclonal antibody that binds VEGF, preventing it from activating its receptors, especially VEGFR2, abrogating subsequent biologic effects. This drug has shown benefit in colorectal, non-small cell lung, and breast cancers, and is approved by the Food and Drug Administration for these indications.
  • Several studies have now evaluated the combination of bevacizumab and the chemotherapeutic agent irinotecan in recurrent malignant gliomas and the results have been more encouraging.
  • the combination of bevacizumab and irinotecan produced a response rate of 67% and 6M-PFS of 56% in recurrent anaplastic gliomas, and a response rate of 57% and a 6M-PFS of 46% in recurrent glioblastomas.
  • VEGF-Trap Another agent proposed for the treatment of malignant gliomas is Aflibercept (VEGF-Trap). This is a soluble hybrid receptor, composed of portions of VEGFR-1 and VEGFR-2 fused to an immunoglobulin G1 Fc domain. Like bevacizumab, it is designed to deplete circulating VEGF, but has significantly greater affinity for VEGF than bevacizumab itself.
  • VEGF receptors have been proposed for the treatment of malignant gliomas.
  • cediranib (AZD2171; Recentin) in patients with recurrent glioblastomas
  • response rates in excess of 50% were observed and the 6M-PFS was increased to approximately 25%.
  • inhibitors of VEGFR such as sorafenib (Nexavar), sunitinib (Sutent), vandetanib (ZD6474; Zactima), pazopanib (GW786034), and vatalanib (PTK787) in glioblastomas are also in progress.
  • agents inhibiting other angiogenic pathways have produced less success.
  • Drugs that inhibit PDGF receptors such as imatinib mesylate (Gleevec) were ineffective, due partly to its poor penetration across the blood-brain barrier.
  • Cilengitide a drug that inhibits av ⁇ 3 and av ⁇ 5 integrins has shown modest activity in glioblastomas and studies combining it with other agents are in progress.
  • viral vectors as gene delivery agents has been proposed for the treatment of malignant gliomas.
  • Such viruses may be engineered to produce anticancer activity by expressing transgenes whose products exert a tumoricidal effect.
  • HSV-tk gene therapy has been the pioneering and most commonly used approach, but oncolytic conditionally replicating adenoviruses and herpes simplex virus mutant vectors, p53, interleukins, interferons, and antisense oligonucleotides have also been used.
  • U.S. Pat. No. 5,747,340 teaches use of a murine endothelial cell-specific promoter which shows selectivity towards angiogenic cells.
  • a viral vector for preparation of a medicament for the treatment of a malignant glioma comprising:
  • a viral vector for the treatment of a malignant glioma comprising:
  • a method of treating a malignant glioma in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a viral vector comprising:
  • the promoter is set forth in SEQ ID NO: 12.
  • the promoter is set forth in SEQ ID NO: 13.
  • the viral vector is an adenoviral vector.
  • the adenoviral vector is a non-replicating adenoviral vector.
  • the promoter comprises at least one copy of a sequence set forth in SEQ ID NO: 6.
  • the promoter comprises at least two copies of a sequence set forth in SEQ ID NO: 6.
  • the promoter comprises a sequence as set forth in SEQ ID NO: 7.
  • the promoter comprises a sequence as set forth in SEQ ID NO: 8.
  • the promoter comprises a hypoxia response element (HRE) as set forth in SEQ ID NO: 5.
  • HRE hypoxia response element
  • the viral vector consists of a sequence as set forth in SEQ ID NO: 9 or SEQ ID NO: 10.
  • the malignant glioma is selected from the group consisting of glioblastoma, astrocytoma, oligodendroglioma, and ependymoma, and juvenile pilocystic astrocytoma.
  • the therapeutically effective amount of the nucleic acid construct is about 10 3 to about 10 16 virus particles.
  • the therapeutically effective amount of the nucleic acid construct is about 10 5 to about 10 13 virus particles.
  • the therapeutically effective amount of the nucleic acid construct is about 10 7 to about 10 12 virus particles.
  • the therapeutically effective amount of the nucleic acid construct is about 1 ⁇ 10 12 to about 5 ⁇ 10 12 virus particles.
  • the therapeutically effective amount of the nucleic acid construct is about 1 ⁇ 10 13 to about 5 ⁇ 10 13 virus particles.
  • the administering comprises intravenous administration.
  • the administering comprises local administration.
  • the administering is in at least two, or at least three or more doses of said viral vector.
  • FIG. 1 is a time chart illustrating an exemplary treatment schedule to measure the in-vivo effects of VB-111 in nude rats pre-inoculated with U87 tumor cells.
  • FIG. 2 is a graph illustrating the effect of VB-111 on survival of rats pre-inoculated with U87 tumor cells.
  • FIG. 3 is a graph illustrating the effect of VB-111 on tumor size (as measured by luciferase activity) of rats pre-inoculated with U87 tumor cells.
  • FIG. 4 is a graph illustrating the effect of VB-111 on tumor size (as measured by MRI) of rats pre-inoculated with U87 tumor cells.
  • FIGS. 5A-F are photographs of brain slices illustrating the effect of VB-111 on tumor size (as measured by MRI) of rats pre-inoculated with U87 tumor cells.
  • the present invention in some embodiments thereof, relates to compositions and methods for treating malignant gliomas and, more particularly, but not exclusively, for treating Glioblastoma multiforme (GBM).
  • GBM Glioblastoma multiforme
  • gliomas the most common subtype of primary brain tumors, are aggressive, highly invasive, and neurologically destructive tumors. These tumors are considered to be among the deadliest of all human cancers. In its most aggressive form, glioblastoma (GBM), median survival ranges from 9 to 12 months. Despite several decades of technological advances in neurosurgery and radiation therapy there has been no significant change in the overall statistics.
  • the present inventors surprisingly found that treatment of glioblastomas in animals could be carried out effectively using viral vectors encoding a toxic molecule (Fas-chimera (Fas-c)) under the control of a promoter which directs transcription in endothelial cells.
  • as-chimera Fas-c
  • the present inventors Using an animal model of glioblastoma whereby rats were pre-inoculated with U87 tumor cells, the present inventors showed that administration of such viral vectors decreased the size of the luciferase-tagged tumors as measured by luciferase activity ( FIG. 3 ) and MRI ( FIGS. 4 and 5 ). The present inventors further showed that administration of such viral vectors increased survival of the rats ( FIG. 2 ).
  • a method of treating a malignant glioma in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a viral vector comprising:
  • malignant glioma refers to a primary central nervous system tumor typically characterized by over-expression of growth factor receptors and/or tumor associated antigens. According to one embodiment the malignant glioma exhibits epidermal growth factor receptor (EGFR) overexpression. According to one embodiment the malignant glioma exhibits platelet-derived growth factor receptor (PDFR) overexpression. According to another embodiment the malignant glioma exhibits both platelet-derived growth factor receptor (PDFR) overexpression and epidermal growth factor receptor (EGFR) overexpression.
  • EGFR epidermal growth factor receptor
  • PDFR platelet-derived growth factor receptor
  • malignant gliomas include, but are not limited to glioblastoma, astrocytoma, oligodendroglioma, ependymoma; and juvenile onset neoplasms such as juvenile pilocystic astrocytoma.
  • Contemplated subjects to be treated include mammals—e.g. humans.
  • the subject has received a prior treatment for the malignant glioma (e.g. radiotherapy and/or chemotherapy) and the malignant glioma has relapsed.
  • the subject has not received a prior treatment for the malignant glioma.
  • viral vector refers to a replication competent or replication-deficient viral particle which are capable of transferring nucleic acid molecules into a host.
  • the present inventors contemplate use of Replication Defective Vectors and Replication Defective Vector-Producing Packaging Cells.
  • examples of such vectors are adenoviral vectors, AAV vectors and retroviral vectors and others described in Shir et al, Cellular and Molecular Neurobiology, Vol. 21, No. 6, December 2001, the contents of which are incorporated herein by reference.
  • virus refers to any of the obligate intracellular parasites having no protein-synthesizing or energy-generating mechanism.
  • the viral genome may be RNA or DNA contained with a coated structure of protein of a lipid membrane.
  • viruses useful in the practice of the present invention include baculoviridiae, parvoviridiae, picomoviridiae, herepesviridiae, poxyiridiae, adenoviridiae, picotmaviridiae.
  • the term recombinant virus includes chimeric (or even multimeric) viruses, i.e. vectors constructed using complementary coding sequences from more than one viral subtype. (See, e.g. Feng, et al.
  • adenovirus is synonymous with the term “adenoviral vector” and refers to viruses of the genus adenoviridiae.
  • adenoviridiae refers collectively to animal adenoviruses of the genus mastadenovirus including but no limited to human, bovine, ovine, equine, canine, porcine, murine and simian adenovirus subgenera.
  • human adenoviruses includes the A-F subgenera as well as the individual serotypes thereof the individual serotypes and A-F subgenera including but not limited to human adenovirus types 1, 2, 3, 4, 4a, 5, 6, 7, 8, 9, 10, 11 (Ad11A and Ad 11P), 12, 13, 14, 15, 16, 17, 18, 19, 19a, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 34a, 35, 35p, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, and 91.
  • bovine adenoviruses includes but is not limited to bovine adenovirus types 1, 2, 3, 4, 7, and 10.
  • canine adenoviruses includes but is not limited to canine types 1 (strains CLL, Glaxo, RI261, Utrect, Toronto 26-61) and 2.
  • equine adenoviruses includes but is not limited to equine types 1 and 2.
  • porcine adenoviruses includes but is not limited to porcine types 3 and 4.
  • the adenovirus is derived from the human adenovirus serotypes 2 or 5.
  • adenovirus vectors can be replication-competent or replication deficient in a target cell.
  • the adenovirus vectors are conditionally or selectively replicating adenoviruses, wherein a gene[s] required for viral replication is [are] operatively linked to a cell and/or context-specific promoter.
  • a gene[s] required for viral replication is [are] operatively linked to a cell and/or context-specific promoter.
  • selectively replicating or conditionally replicating viral vectors are known in the art (see, for example, U.S. Pat. No. 7,691,370).
  • the adenovirus vector is a conditionally replicating adenovirus wherein the E1 gene is under transcriptional control of the pre-proendothelin promoter PPE-1 (PPE-1, SEQ ID NO: 13).
  • the adenovirus vector is a conditionally replicating or selectively replicating adenovirus wherein the E1 gene is under transcriptional control of the modified pre-proendothelin promoter PPE-1-3X (PPE-1-3X, SEQ ID NO: 12).
  • adenovirus vectors suitable for use with the present invention include all adenovirus serotypes having hexon protein structure.
  • Viral vectors suitable for therapeutic use include adenoviral vectors, retrovirusal vectors, AAV, herpesvirus vectors and the like. Engineering and production of viral vectors is well known in the art, as described in detail in, for example, U.S. Pat. No.
  • the adenovirus is a C-type adenovirus (Ad5, Ad2), a B-type adenovirus (Ad3, Ad16, Ad21, Ad35, Ad50), an E-type adenovirus (Ad4) or an F-type adenovirus (Ad41).
  • adenoviral vector refers to a vector in which, among the nucleic acid molecules in the viral particle, sequences necessary to function as a viral vector are based on the adenoviral genome.
  • the adenoviral vector is a non-replicating serotype 5 (Ad5) adenoviral vector.
  • the adenoviral vector comprises a sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 11.
  • oncolytic viruses which reproduce themselves in cancer cells and subsequently kill the initially infected cells by lysis. Such viruses proceed to infect adjacent cells thus repeating the cycle.
  • oncolytic viruses include, but are not limited to Herpes Simplex Virus, conditionally replicative Ads (CRAds) and reoviruses.
  • CRAd vectors Two major strategies for development of CRAd vectors have been developed, mainly focusing on the genetic engineering of the early 1 (E1) genes to restrict virus replication to target cells and to spare normal tissue.
  • E1A immediately early
  • E1B early adenoviral region
  • virus replication is controlled via a tumor/tissue-specific promoter.
  • Reovirus is a naturally occurring oncolytic virus that requires activated Ras signaling pathways of tumor cells for its replication. Ras pathways are activated in most malignant gliomas via upstream signaling by receptor tyrosine kinases.
  • viral vectors of this aspect of the present invention comprise:
  • viral vectors are constructed using genetic recombination technology—i.e. recombinant viral vectors.
  • the Fas-chimera (Fas-c), is a previously described fusion of two “death receptors”, constructed from the extracellular region of TNFR1 (SEQ ID NO: 2) and the trans-membrane and intracellular regions of Fas (SEQ ID NO: 3) [Boldin M P et al. J Biol Chem (1995) 270(14):7795-8].
  • the Fas-c is encoded by a polynucleotide as set forth in SEQ ID NO: 4.
  • a viral construct e.g. an adenoviral construct
  • an endothelial/periendothelial cell-specific promoter operatively linked to other cytotoxic polypeptides for the treatment of malignant glioma.
  • polypeptides include but are not limited to suicide polypeptides such as p53 and egr-1-TNF-alpha, cytotoxic pro-drug/enzymes for drug susceptibility therapy such as ganciclovir/thymidine kinase and 5-fluorocytosine/cytosine deaminase, and antimetastatic polypeptides such as 5 E1A.
  • suicide polypeptides such as p53 and egr-1-TNF-alpha
  • cytotoxic pro-drug/enzymes for drug susceptibility therapy such as ganciclovir/thymidine kinase and 5-fluorocytosine/cytosine deaminase
  • antimetastatic polypeptides such as 5 E1A.
  • promoter refers to a DNA sequence which directs transcription of a polynucleotide sequence operatively linked thereto in the cell in a constitutive or inducible manner.
  • the promoter may also comprise enhancer elements which stimulate transcription from the linked promoter.
  • endothelial cell-specific promoter refers to a promoter which directs expression of a gene operatively linked thereto in endothelial cells, wherein the level of expression in endothelial cells is at least 2 times higher than in non-endothelial cells. According to a particular embodiment, the level of expression in endothelial cells is at least 5 times higher than in non-endothelial cells.
  • periendothelial cell-specific promoter refers to a promoter which directs expression of a gene operatively linked thereto in periendothelial cells (i.e., pericytes in small vessels or smooth muscle cells in larger vessels), wherein the level of expression in endothelial cells is at least 2 times higher than in non-periendothelial cells. According to a particular embodiment, the level of expression in periendothelial cells is at least 5 times higher than in non-periendothelial cells.
  • Exemplary endothelial cell-specific promoters or periendothelial cell-specific promoters include, but are not limited to the preproendothelin-1 (PPE-1) promoter, and modifications thereof such as described herein below, the TIE-1 promoter, the TIE-2 promoter, the Endoglin promoter, the von Willerband promoter, the KDR/flk-1 promoter, The FLT-1 promoter, the Egr-1 promoter, the ICAM-1 promoter, the VCAM-1 promoter, the PECAM-1 promoter and the aortic carboxypeptidase-like protein (ACLP) promoter.
  • PPE-1 preproendothelin-1
  • the preproendothelial promoter refers to the preproendothelin-1 promoter, of mammalian origin.
  • the preproendothelin 1 promoter is a murine preproendothelin 1 promoter as set forth in SEQ ID NO: 13.
  • the promoter comprises at least one copy of an enhancer element that confers endothelial cell specific transcriptional activity.
  • the enhancer element is naturally found positioned between the ⁇ 364 bp and ⁇ 320 bp of the murine PPE-1 promoter (as set forth in SEQ ID NO: 6).
  • the promoter comprises at least two and more preferably three of the above described enhancer elements.
  • the promoter comprises two of the above described enhancer elements on one strand of the promoter DNA and one of the above described enhancer element on the complementary strand of the promoter DNA (as set forth in SEQ ID NO:7).
  • the promoter further comprises at least one hypoxia response element—e.g. comprising a sequence as set forth in SEQ ID NO: 5.
  • An exemplary promoter which can be used in the context of the present invention comprises a sequence as set forth in SEQ ID NO: 12.
  • This promoter is also referred to herein as the PPE-1-3x promoter.
  • This sequence comprises SEQ ID NO: 5 and SEQ ID NO: 7 (which itself comprises two copies of SEQ ID NO: 6 either side of one copy of SEQ ID NO: 8).
  • the viral vector consists of a sequence as set forth in SEQ ID NOs: 9 or 10.
  • This viral vector is also referred to herein as VB111 and AD5PPE-1-3X-fas-chimera.
  • This sequence comprises SEQ ID NO: 12 in the antisense orientation at position 460-1437.
  • This sequence also comprises SEQ ID NO: 7 in the antisense orientation at position 894-1036; a single copy of SEQ ID NO: 8 in the antisense orientation at position 951-997; a first copy of SEQ ID NO: 6 in the antisense orientation at position 907-950; a second copy of SEQ ID NO: 6 in the antisense orientation at position 993-1036; and a third copy of SEQ ID NO: 6 at position 823-866 in the sense orientation.
  • the viral vector comprises additional polynucleotide sequences capable of enhancing or inhibiting transcriptional activity of an endothelial specific promoter.
  • the additional polynucleotide sequence includes an isolated polynucleotide comprising at least 6 nucleotides of element X of a pre-proendothelin (PPE-1) promoter, the element X having a wild type sequence as set forth by SEQ ID NO:6, wherein the at least 6 nucleotides comprise at least 2 consecutive sequences derived from SEQ ID NO:6, each of the at least 2 consecutive sequences comprises at least 3 nucleotides, at least one of the at least 3 nucleotide being positioned next to at least one nucleotide position in SEQ ID NO:6, the at least one nucleotide position in SEQ ID NO:6 is selected from the group consisting of:
  • the at least one nucleotide position is mutated as compared to SEQ ID NO:6 by at least one nucleotide substitution, at least one nucleotide deletion and/or at least one nucleotide insertion, with the proviso that a mutation of the at least one nucleotide position does not result in nucleotides GGTA at position 21-24 of SEQ ID NO:6 and/or in nucleotides CATG at position 29-32 of SEQ ID NO:6, such that when the isolated polynucleotide is integrated into the PPE-1 promoter and placed upstream of a reporter gene (e.g., luciferase coding sequence) the expression level of the reporter gene is upregulated or downregulated as compared to when SEQ ID NO:6 is similarly integrated into the PPE-1 promoter and placed upstream of the reporter gene coding sequence.
  • a reporter gene e.g., luciferase coding sequence
  • the isolated polynucleotide is not naturally occurring in a genome or a whole chromosome sequence of an organism.
  • the at least 6 nucleotides of element X comprise at least 2 consecutive sequences derived from SEQ ID NO:6.
  • sequence derived from SEQ ID NO:6 refers to a nucleic acid sequence (a polynucleotide) in which the nucleotides appear in the same order as in the nucleic acid sequence of SEQ ID NO:6 from which they are derived. It should be noted that the order of nucleotides is determined by the chemical bond (phosphodiester bond) formed between a 3′-OH of a preceding nucleotide and the 5′-phosphate of the following nucleotide.
  • each of the at least 2 consecutive sequences comprises at least 3 nucleotides, e.g., 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, 30 nucleotide, 31 nucleotides, 32 nucleotides, 33 nucleotides, 34 nucleotides, 10 nucleot
  • the isolated polynucleotide comprises at least 2 consecutive sequences derived from SEQ ID NO:6. According to some embodiments of the invention, the isolated polynucleotide comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 consecutive sequences derived from SEQ ID NO:6.
  • wild type refers to the nucleic acid sequence as appears in SEQ ID NO:6.
  • examples include, but are not limited to wild type M4 sequence (SEQ ID NO: 15), wild type M5 sequence (SEQ ID NO: 16), wild type M8 (SEQ ID NO:19), wild type M6 sequence (SEQ ID NO:17), wild type M7 sequence (SEQ ID NO:18), wild type M1 (SEQ ID NO:20) and wild type M3 sequence (SEQ ID NO:21).
  • the mutation is an insertion of at least one nucleotide in a nucleotide position with respect to SEQ ID NO:6.
  • the insertion includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 nucleotides, e.g., at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, at least about 100, at least about 200, at least about 300, or more nucleotides.
  • sequence which is inserted by the mutation can be derived from any source (e.g., species, tissue or cell type), and is not limited to the source of the sequence of element X.
  • the mutation is a combination of any of the mutation types described above, i.e., substitution, insertion and deletion.
  • substitution i.e., substitution, insertion and deletion.
  • another nucleotide position in SEQ ID NO:6 can be subject to a deletion or insertion.
  • another nucleotide position in SEQ ID NO:6 can be subject to a substitution or insertion.
  • nucleotide position in SEQ ID NO:6 can be subject to an insertion mutation
  • another nucleotide position in SEQ ID NO:6 can be subject to a substitution or deletion. It should be noted that various other combinations are possible.
  • the mutation in the isolated polynucleotide of the invention does not result in nucleotides GGTA at position 21-24 of SEQ ID NO:6 and/or in nucleotides CATG at position 29-32 of SEQ ID NO:6.
  • the phrase “integrated into the PPE-1 promoter” refers to a nucleotide sequence (the isolated polynucleotide) which is covalently conjugated within the PPE-1 promoter sequence.
  • the isolated polynucleotide further comprises at least one copy of a nucleic acid sequence selected from the group consisting of:
  • the isolated polynucleotide is integrated into (within), downstream of, or upstream of any known (or unknown) promoter sequence to thereby regulate (e.g., increase, decrease, modulate tissue-specificity, modulate inductive or constitutive expression) the transcriptional promoting activity of the promoter.
  • the isolated polynucleotide is for increasing expression of a heterologous polynucleotide operably linked thereto in endothelial cells.
  • a polynucleotide can include wild type sequences of M4 and/or M5 in the presence or absence of additional sequences from element X, and/or in the presence of other mutated sequences from element X.
  • the isolated polynucleotide comprises at least one copy of the wild type M4 sequence set forth by SEQ ID NO: 15 (CATTC).
  • the isolated polynucleotide comprises at least one copy of the wild type M5 sequence set forth by SEQ ID NO: 16 (CAATG).
  • the isolated polynucleotide comprises at least one copy of the wild type M4 sequence set forth by SEQ ID NO: 15 (CATTC) and at least one copy of the wild type M5 sequence set forth by SEQ ID NO: 16 (CAATG).
  • the at least one nucleotide position which is mutated as compared to SEQ ID NO:6 is at least one nucleotide of the wild type M8 sequence set forth by SEQ ID NO: 19 (GCTTC).
  • GCTTC wild type M8 sequence set forth by SEQ ID NO: 19
  • an isolated polynucleotide may further include a wild type M6 sequence (SEQ ID NO:17) and/or a wild type M7 sequence (SEQ ID NO:18)
  • Non-limiting examples of isolated polynucleotides which include at least one copy of the wild type M4 sequence set forth by SEQ ID NO: 15 (CATTC) and a mutation in at least one nucleotide of the wild type M8 sequence set forth by SEQ ID NO: 19 (GCTTC) are provided in SEQ ID NOs:55-62.
  • Non-limiting examples of isolated polynucleotides which include at least one copy of the wild type M5 sequence set forth by SEQ ID NO: 16 (CAATG) and a mutation in at least one nucleotide of the wild type M8 sequence set forth by SEQ ID NO: 19 (GCTTC) are provided in SEQ ID NOs: 63-66.
  • Non-limiting examples of isolated polynucleotides which include at least one copy of the wild type M4 sequence set forth by SEQ ID NO: 15 (CATTC), at least one copy of the wild type M5 sequence set forth by SEQ ID NO: 16 (CAATG) and a mutation in at least one nucleotide of the wild type M8 sequence set forth by SEQ ID NO: 19 (GCTTC) are provided in SEQ ID NOs: 67-70.
  • the isolated polynucleotide further comprising at least one copy of wild type M1 sequence set forth by SEQ ID NO: (GTACT).
  • Non-limiting examples of isolated polynucleotides which include at least one copy of the wild type M4 sequence set forth by SEQ ID NO: 15 (CATTC), at least one copy of the wild type M1 sequence set forth by SEQ ID NO: 20 (GTACT), and a mutation in at least one nucleotide of the wild type M8 sequence set forth by SEQ ID NO: 19 (GCTTC) are provided in SEQ ID NOs: 71-105.
  • Non-limiting examples of isolated polynucleotides which include at least one copy of the wild type M5 sequence set forth by SEQ ID NO: 16 (CAATG), at least one copy of the wild type M1 sequence set forth by SEQ ID NO: 20 (GTACT) and a mutation in at least one nucleotide of the wild type M8 sequence set forth by SEQ ID NO: 19 (GCTTC) are provided in SEQ ID NOs: 106-136.
  • Non-limiting examples of isolated polynucleotides which include at least one copy of the wild type M4 sequence set forth by SEQ ID NO: 15 (CATTC), at least one copy of the wild type M5 sequence set forth by SEQ ID NO: 16 (CAATG), at least one copy of the wild type M1 sequence set forth by SEQ ID NO: 20 (GTACT) and a mutation in at least one nucleotide of the wild type M8 sequence set forth by SEQ ID NO: 19 (GCTTC) are provided in SEQ ID NOs:137-152.
  • the isolated polynucleotide reduces expression of a heterologous polynucleotide operably linked thereto in endothelial cells.
  • a polynucleotide can include mutations in M4 and/or M5 in the presence or absence of additional sequences from element X, and/or in the presence of other mutated sequences from element X.
  • the at least one nucleotide position which is mutated as compared to SEQ ID NO:6 is at least one nucleotide of the wild type M4 sequence set forth by SEQ ID NO: 15 (CATTC).
  • Non-limiting examples of isolated polynucleotides which includes a mutation in at least one nucleotide of the wild type M4 sequence set forth by SEQ ID NO:46 are provided in SEQ ID NOs:153-162.
  • the at least one nucleotide position which is mutated as compared to SEQ ID NO:6 is at least one nucleotide of the wild type M5 sequence set forth by SEQ ID NO: 16 (CAATG).
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M5 sequence set forth by SEQ ID NO: 16 are provided in SEQ ID NOs:163-171.
  • the at least one nucleotide position which is mutated as compared to SEQ ID NO:6 is at least one nucleotide of the wild type M4 sequence set forth by SEQ ID NO: 15 (CATTC) and at least one nucleotide of the wild type M5 sequence set forth by SEQ ID NO: 16 (CAATG).
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M4 sequence set forth by SEQ ID NO: 15 (CATTC) and a mutation in at least one nucleotide of the wild type M5 sequence set forth by SEQ ID NO: 16 (CAATG) are provided in SEQ ID NOs:172-180.
  • the isolated polynucleotide is for increasing expression of a heterologous polynucleotide operably linked thereto in cells other than endothelial cells.
  • a polynucleotide can include mutations in M4 and/or M5 and wild type sequences of M6 and/or M7, in the presence or absence of additional sequences from element X, and/or in the presence of other mutated sequences from element X.
  • the isolated polynucleotide comprises a mutation in M4 (SEQ ID NO: 15) and/or in M5 (SEQ ID NO: 16) and at least one copy of the wild type M6 set forth by SEQ ID NO: 17 (GGGTG) and/or at least one copy of wild type M7 set forth by SEQ ID NO:18.
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M4 sequence set forth by SEQ ID NO: 15 (CATTC) and at least one copy of the wild type M6 set forth by SEQ ID NO: 17 (GGGTG) are provided in SEQ ID NOs:181-182.
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M5 sequence set forth by SEQ ID NO: 16 (CAATG) and at least one copy of the wild type M6 set forth by SEQ ID NO: 17 (GGGTG) are provided in SEQ ID NOs:183-189.
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M4 sequence set forth by SEQ ID NO: 15 (CATTC), a mutation in at least one nucleotide of the wild type M5 sequence set forth by SEQ ID NO: 16 (CAATG) and at least one copy of the wild type M6 set forth by SEQ ID NO: 17 (GGGTG) are provided in SEQ ID NOs:190-191.
  • the isolated polynucleotide further comprises at least one copy of the wild type M7 sequence set forth by SEQ ID NO: 18 (ACTTT).
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M4 sequence set forth by SEQ ID NO: 15 (CATTC) and at least one copy of the wild type M7 sequence set forth by SEQ ID NO: 18 (ACTTT) are provided in SEQ ID NOs:192-195.
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M5 sequence set forth by SEQ ID NO: 16 (CAATG) and at least one copy of the wild type M7 sequence set forth by SEQ ID NO: 18 (ACTTT) are provided in SEQ ID NOs:196-198.
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M4 sequence set forth by SEQ ID NO: 15 CATTC
  • CATTC a mutation in at least one nucleotide of the wild type M5 sequence set forth by SEQ ID NO: 16
  • CAATG a mutation in at least one nucleotide of the wild type M5 sequence set forth by SEQ ID NO: 16
  • ACTTT at least one copy of the wild type M7 sequence set forth by SEQ ID NO: 18
  • the isolated polynucleotide further comprises at least one copy of the wild type M6 set forth by SEQ ID NO: 17 (GGGTG) and at least one copy of the wild type M7 sequence set forth by SEQ ID NO: 18 (ACTTT).
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M4 sequence set forth by SEQ ID NO: 15 (CATTC), at least one, copy of the wild type M6 set forth by SEQ ID NO: 17 (GGGTG) and at least one copy of the wild type M7 sequence set forth by SEQ ID NO: 18 (ACTTT) are provided in SEQ ID NOs:203-205.
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M5 sequence set forth by SEQ ID NO: 16 (CAATG), at least one copy of the wild type M6 set forth by SEQ ID NO: 17 (GGGTG) and at least one copy of the wild type M7 sequence set forth by SEQ ID NO: 18 (ACTTT) are provided in SEQ ID NOs:206-207.
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M4 sequence set forth by SEQ ID NO: 15 (CATTC), a mutation in at least one nucleotide of the wild type M5 sequence set forth by SEQ ID NO: 16 (CAATG), at least one copy of the wild type M6 set forth by SEQ ID NO: 17 (GGGTG) and at least one copy of the wild type M7 sequence set forth by SEQ ID NO: 18 (ACTTT) are provided in SEQ ID NOs:208-209.
  • the isolated polynucleotide reduces expression in cells of a heterologous polynucleotide operably linked thereto.
  • a polynucleotide can include mutations in M4, M5, M6 and/or M7, in the presence or absence of additional sequences from element X, and/or in the presence of other mutated sequences from element X.
  • the isolated polynucleotide comprises at least one mutation in wild type M4 (SEQ ID NO: 15) and/or in wild type M5 (SEQ ID NO:47) and in wild type M6 set forth by SEQ ID NO: 17 (GGGTG).
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M4 sequence set forth by SEQ ID NO: 15 (CATTC) and a mutation in at least one nucleotide position of the wild type M6 set forth by SEQ ID NO: 17 (GGGTG) are provided in SEQ ID NOs:210-213.
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M5 sequence set forth by SEQ ID NO: 16 (CAATG) and a mutation in at least one nucleotide position of the wild type M6 set forth by SEQ ID NO: 17 (GGGTG) are provided in SEQ ID NOs:214-222.
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M4 sequence set forth by SEQ ID NO: 15 (CATTC), a mutation in at least one nucleotide of the wild type M5 sequence set forth by SEQ ID NO: 16 (CAATG), and a mutation in at least one nucleotide position of the wild type M6 set forth by SEQ ID NO: 17 (GGGTG) are provided in SEQ ID NOs:223-231.
  • the isolated polynucleotide further comprises at least one mutation in wild type M7 set forth by SEQ ID NO: 18 (ACTTT).
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M4 sequence set forth by SEQ ID NO: 15 (CATTC) and a mutation in at least one nucleotide position of the wild type M7 set forth by SEQ ID NO: 18 (ACTTT) are provided in SEQ ID NOs:232-236.
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M5 sequence set forth by SEQ ID NO: 16 (CAATG) and a mutation in at least one nucleotide position of the wild type M7 set forth by SEQ ID NO: 18 (ACTTT) are provided in SEQ ID NOs:237-240.
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M4 sequence set forth by SEQ ID NO: 15 (CATTC), a mutation in at least one nucleotide of the wild type M5 sequence set forth by SEQ ID NO: 16 (CAATG), and a mutation in at least one nucleotide position of the wild type M7 set forth by SEQ ID NO: 18 (ACTTT) are provided in SEQ ID NOs:241-248.
  • CATTC CATTC
  • CAATG a mutation in at least one nucleotide of the wild type M5 sequence set forth by SEQ ID NO: 16
  • ACTTT a mutation in at least one nucleotide position of the wild type M7 set forth by SEQ ID NO: 18
  • the isolated polynucleotide further comprises at least one mutation in wild type M6 set forth by SEQ ID NO: 17 (GGGTG) and at least one mutation in wild type M7 set forth by SEQ ID NO: 18 (ACTTT).
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M4 sequence set forth by SEQ ID NO: 15 (CATTC), a mutation in at least one nucleotide position of the wild type M6 set forth by SEQ ID NO: 17 (GGGTG) and a mutation in at least one nucleotide position of the wild type M7 set forth by SEQ ID NO: 18 (ACTTT) are provided in SEQ ID NOs:249-258.
  • CATTC CATTC
  • GGGTG a mutation in at least one nucleotide position of the wild type M6 set forth by SEQ ID NO: 17
  • ACTTT a mutation in at least one nucleotide position of the wild type M7 set forth by SEQ ID NO: 18
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M5 sequence set forth by SEQ ID NO: 16 (CAATG), a mutation in at least one nucleotide position of the wild type M6 set forth by SEQ ID NO: 17 (GGGTG) and a mutation in at least one nucleotide position of the wild type M7 set forth by SEQ ID NO: 18 (ACTTT) are provided in SEQ ID NOs:259-264.
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M4 sequence set forth by SEQ ID NO: 15 (CATTC), a mutation in at least one nucleotide of the wild type M5 sequence set forth by SEQ ID NO: 16 (CAATG), a mutation in at least one nucleotide position of the wild type M6 set forth by SEQ ID NO: 17 (GGGTG) and a mutation in at least one nucleotide position of the wild type M7 set forth by SEQ ID NO: 18 (ACTTT) are provided in SEQ ID NOs:265-270.
  • CATTC CATTC
  • CAATG a mutation in at least one nucleotide of the wild type M5 sequence set forth by SEQ ID NO: 16
  • GGGTG a mutation in at least one nucleotide position of the wild type M6 set forth by SEQ ID NO: 17
  • ACTTT a mutation in at least one nucleotide position of the
  • the isolated polynucleotide comprises at least one copy of the wild type M8 sequence set forth by SEQ ID NO: 19 (GCTTC) with additional wild type or mutated sequences derived from element X (SEQ ID NO:6).
  • Non-limiting examples of isolated polynucleotides which includes a mutation in at least one nucleotide of the wild type M4 sequence set forth by SEQ ID NO: 15 (CATTC) and at least one copy of the wild type M8 sequence set forth by SEQ ID NO: 19 (GCTTC) are provided in SEQ ID NOs:271-279.
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M5 sequence set forth by SEQ ID NO: 16 (CAATG) and at least one copy of the wild type M8 sequence set forth by SEQ ID NO: 19 (GCTTC) are provided in SEQ ID NOs:280-287.
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M4 sequence set forth by SEQ ID NO: 15 CATTC
  • CATTC a mutation in at least one nucleotide of the wild type M5 sequence set forth by SEQ ID NO: 16
  • GCTTC at least one copy of the wild type M8 sequence set forth by SEQ ID NO: 19
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M4 sequence set forth by SEQ ID NO: 15 (CATTC), at least one copy of the wild type M6 set forth by SEQ ID NO: 17 (GGGTG) and at least one copy of the wild type M8 sequence set forth by SEQ ID NO: 19 (GCTTC) are provided in SEQ ID NOs:294-298.
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M5 sequence set forth by SEQ ID NO: 16 (CAATG), at least one copy of the wild type M6 set forth by SEQ ID NO: 17 (GGGTG) and at least one copy of the wild type M8 sequence set forth by SEQ ID NO: 19 (GCTTC) are provided in SEQ ID NOs:299-301.
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M4 sequence set forth by SEQ ID NO: 15 (CATTC), a mutation in at least one nucleotide of the wild type M5 sequence set forth by SEQ ID NO: 16 (CAATG), at least one copy of the wild type M6 set forth by SEQ ID NO: 17 (GGGTG) and at least one copy of the wild type M8 sequence set forth by SEQ ID NO: 19 (GCTTC) are provided in SEQ ID NOs:302-303.
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M4 sequence set forth by SEQ ID NO: 15 (CATTC), at least one copy of the wild type M7 sequence set forth by SEQ ID NO: 18 (ACTTT) and at least one copy of the wild type M8 sequence set forth by SEQ ID NO: 19 (GCTTC) are provided in SEQ ID NOs:304-308.
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M5 sequence set forth by SEQ ID NO: 16 CAATG
  • at least one copy of the wild type M7 sequence set forth by SEQ ID NO: 18 ACTTT
  • at least one copy of the wild type M8 sequence set forth by SEQ ID NO: 19 GCTTC
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M4 sequence set forth by SEQ ID NO: 15 (CATTC), a mutation in at least one nucleotide of the wild type M5 sequence set forth by SEQ ID NO: 16 (CAATG), at least one copy of the wild type M7 sequence set forth by SEQ ID NO: 18 (ACTTT) and at least one copy of the wild type M8 sequence set forth by SEQ ID NO: 19 (GCTTC) are provided in SEQ ID NOs:312-315.
  • CATTC CATTC
  • CAATG a mutation in at least one nucleotide of the wild type M5 sequence set forth by SEQ ID NO: 16
  • ACTTT at least one copy of the wild type M7 sequence set forth by SEQ ID NO: 18
  • GCTTC at least one copy of the wild type M8 sequence set forth by SEQ ID NO: 19
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M4 sequence set forth by SEQ ID NO: 15 (CATTC), at least one copy of the wild type M6 set forth by SEQ ID NO: 17 (GGGTG), at least one copy of the wild type M7 sequence set forth by SEQ ID NO: 18 (ACTTT) and at least one copy of the wild type M8 sequence set forth by SEQ ID NO: 19 (GCTTC) are provided in SEQ ID NO:316.
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M5 sequence set forth by SEQ ID NO: 16 (CAATG), at least one copy of the wild type M6 set forth by SEQ ID NO: 17 (GGGTG), at least one copy of the wild type M7 sequence set forth by SEQ ID NO: 18 (ACTTT) and at least one copy of the wild type M8 sequence set forth by SEQ ID NO: 19 (GCTTC) are provided in SEQ ID NO:317.
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M4 sequence set forth by SEQ ID NO: 15 (CATTC), a mutation in at least one nucleotide of the wild type M5 sequence set forth by SEQ ID NO: 16 (CAATG), at least one copy of the wild type M6 set forth by SEQ ID NO: 17 (GGGTG), at least one copy of the wild type M7 sequence set forth by SEQ ID NO: 18 (ACTTT) and at least one copy of the wild type M8 sequence set forth by SEQ ID NO: 19 (GCTTC) are provided in SEQ ID NO:318.
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M4 sequence set forth by SEQ ID NO: 15 (CATTC), a mutation in at least one nucleotide position of the wild type M6 set forth by SEQ ID NO: 17 (GGGTG) and at least one copy of the wild type M8 sequence set forth by SEQ ID NO: 19 (GCTTC) are provided in SEQ ID NOs:319-327.
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M5 sequence set forth by SEQ ID NO: 16 (CAATG), a mutation in at least one nucleotide position of the wild type M6 set forth by SEQ ID NO: 17 (GGGTG) and at least one copy of the wild type M8 sequence set forth by SEQ ID NO: 19 (GCTTC) are provided in SEQ ID NOs:328-333.
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M4 sequence set forth by SEQ ID NO: 15 (CATTC), a mutation in at least one nucleotide of the wild type M5 sequence set forth by SEQ ID NO: 16 (CAATG), a mutation in at least one nucleotide position of the wild type M6 set forth by SEQ ID NO: 17 (GGGTG) and at least one copy of the wild type M8 sequence set forth by SEQ ID NO: 19 (GCTTC) are provided in SEQ ID NOs:334-337.
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M4 sequence set forth by SEQ ID NO: 15 (CATTC), a mutation in at least one nucleotide position of the wild type M7 set forth by SEQ ID NO: 18 (ACTTT) and at least one copy of the wild type M8 sequence set forth by SEQ ID NO: 19 (GCTTC) are provided in SEQ ID NOs:338-344.
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M5 sequence set forth by SEQ ID NO: 16 CAATG
  • ACTTT a mutation in at least one nucleotide position of the wild type M7 set forth by SEQ ID NO: 18
  • GCTTC at least one copy of the wild type M8 sequence set forth by SEQ ID NO: 19
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M4 sequence set forth by SEQ ID NO: 15 (CATTC), a mutation in at least one nucleotide of the wild type M5 sequence set forth by SEQ ID NO: 16 (CAATG), a mutation in at least one nucleotide position of the wild type M7 set forth by SEQ ID NO: 18 (ACTTT) and at least one copy of the wild type M8 sequence set forth by SEQ ID NO: 19 (GCTTC) are provided in SEQ ID NOs:349-354.
  • CATTC CATTC
  • CAATG a mutation in at least one nucleotide position of the wild type M7 set forth by SEQ ID NO: 18
  • GCTTC at least one copy of the wild type M8 sequence set forth by SEQ ID NO: 19
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M4 sequence set forth by SEQ ID NO: 15 (CATTC), a mutation in at least one nucleotide position of the wild type M6 set forth by SEQ ID NO: 17 (GGGTG), a mutation in at least one nucleotide position of the wild type M7 set forth by SEQ ID NO: 18 (ACTTT) and at least one copy of the wild type M8 sequence set forth by SEQ ID NO: 19 (GCTTC) are provided in SEQ ID NOs:355-361.
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M5 sequence set forth by SEQ ID NO: 16 (CAATG), a mutation in at least one nucleotide position of the wild type M6 set forth by SEQ ID NO: 17 (GGGTG), a mutation in at least one nucleotide position of the wild type M7 set forth by SEQ ID NO: 18 (ACTTT) and at least one copy of the wild type M8 sequence set forth by SEQ ID NO: 19 (GCTTC) are provided in SEQ ID NOs:362-365.
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M4 sequence set forth by SEQ ID NO: 15 (CATTC), a mutation in at least one nucleotide of the wild type M5 sequence set forth by SEQ ID NO: 16 (CAATG), a mutation in at least one nucleotide position of the wild type M6 set forth by SEQ ID NO: 17 (GGGTG), a mutation in at least one nucleotide position of the wild type M7 set forth by SEQ ID NO: 18 (ACTTT) and at least one copy of the wild type M8 sequence set forth by SEQ ID NO: 19 (GCTTC) are provided in SEQ ID NOs:366-369.
  • the isolated polynucleotide comprises at least one copy of the wild type M3 sequence set forth by SEQ ID NO: 21 (CTTTT) with additional wild type or mutated sequences derived from element X (SEQ ID NO:6).
  • Non-limiting examples of isolated polynucleotides which includes a mutation in at least one nucleotide of the wild type M4 sequence set forth by SEQ ID NO: 15 (CATTC) and at least one copy of the wild type M3 sequence set forth by SEQ ID NO: 21 (CTTTT) are provided in SEQ ID NOs:378-384.
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M5 sequence set forth by SEQ ID NO: 16 (CAATG) and at least one copy of the wild type M3 sequence set forth by SEQ ID NO: 21 (CTTTT) are provided in SEQ ID NOs:628-634.
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M4 sequence set forth by SEQ ID NO: 15 CATTC
  • CATTC a mutation in at least one nucleotide of the wild type M5 sequence set forth by SEQ ID NO: 16
  • CAATG a mutation in at least one nucleotide of the wild type M5 sequence set forth by SEQ ID NO: 16
  • CTTT at least one copy of the wild type M3 sequence set forth by SEQ ID NO: 21
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M4 sequence set forth by SEQ ID NO: 15 (CATTC), at least one copy of the wild type M6 set forth by SEQ ID NO: 17 (GGGTG) and at least one copy of the wild type M3 sequence set forth by SEQ ID NO: 21 (CTTTT) are provided in SEQ ID NOs:385-390.
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M5 sequence set forth by SEQ ID NO: 16 (CAATG), at least one copy of the wild type M6 set forth by SEQ ID NO: 17 (GGGTG) and at least one copy of the wild type M3 sequence set forth by SEQ ID NO: 21 (CTTTT) are provided in SEQ ID NOs:391-396.
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M4 sequence set forth by SEQ ID NO: 15 (CATTC), a mutation in at least one nucleotide of the wild type M5 sequence set forth by SEQ ID NO: 16 (CAATG), at least one copy of the wild type M6 set forth by SEQ ID NO: 17 (GGGTG) and at least one copy of the wild type M3 sequence set forth by SEQ ID NO: 21 (CTTTT) are provided in SEQ ID NOs:397-401.
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M4 sequence set forth by SEQ ID NO: 15 (CATTC), at least one copy of the wild type M7 sequence set forth by SEQ ID NO: 18 (ACM) and at least one copy of the wild type M3 sequence set forth by SEQ ID NO: 21 (CTTTT) are provided in SEQ ID NOs:402-409.
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M5 sequence set forth by SEQ ID NO: 16 (CAATG), at least one copy of the wild type M7 sequence set forth by SEQ ID NO: 18 (ACM) and at least one copy of the wild type M3 sequence set forth by SEQ ID NO: 21 (CTTTT) are provided in SEQ ID NOs:410-417.
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M4 sequence set forth by SEQ ID NO: 15 (CATTC), a mutation in at least one nucleotide of the wild type M5 sequence set forth by SEQ ID NO: 16 (CAATG), at least one copy of the wild type M7 sequence set forth by SEQ ID NO: 18 (ACTTT) and at least one copy of the wild type M3 sequence set forth by SEQ ID NO: 21 (CTTTT) are provided in SEQ ID NOs:418-423.
  • CATTC CATTC
  • CAATG a mutation in at least one nucleotide of the wild type M5 sequence set forth by SEQ ID NO: 16
  • ACTTT at least one copy of the wild type M7 sequence set forth by SEQ ID NO: 18
  • CTTTT at least one copy of the wild type M3 sequence set forth by SEQ ID NO: 21
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M4 sequence set forth by SEQ ID NO: 15 (CATTC), at least one copy of the wild type M6 set forth by SEQ ID NO: 17 (GGGTG), at least one copy of the wild type M7 sequence set forth by SEQ ID NO: 18 (ACTTT) and at least one copy of the wild type M3 sequence set forth by SEQ ID NO: 21 (CTTTT) are provided in SEQ ID NOs:424-425.
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M5 sequence set forth by SEQ ID NO: 16 (CAATG), at least one copy of the wild type M6 set forth by SEQ ID NO: 17 (GGGTG), at least one copy of the wild type M7 sequence set forth by SEQ ID NO: 18 (ACTTT) and at least one copy of the wild type M3 sequence set forth by SEQ ID NO: 21 (CTTTT) are provided in SEQ ID NOs:538-540.
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M4 sequence set forth by SEQ ID NO: 15 (CATTC), a mutation in at least one nucleotide of the wild type M5 sequence set forth by SEQ ID NO: 16 (CAATG), at least one copy of the wild type M6 set forth by SEQ ID NO: 17 (GGGTG), at least one copy of the wild type M7 sequence set forth by SEQ ID NO: 18 (ACTTT) and at least one copy of the wild type M3 sequence set forth by SEQ ID NO: 21 (CTTTT) are provided in SEQ ID NO:426.
  • CATTC CATTC
  • CAATG a mutation in at least one nucleotide of the wild type M5 sequence set forth by SEQ ID NO: 16
  • GGGTG at least one copy of the wild type M6 set forth by SEQ ID NO: 17
  • ACTTT at least one copy of the wild type M7 sequence set forth by SEQ ID NO
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M4 sequence set forth by SEQ ID NO: 15 (CATTC), a mutation in at least one nucleotide position of the wild type M6 set forth by SEQ ID NO: 17 (GGGTG) and at least one copy of the wild type M3 sequence set forth by SEQ ID NO: 21 (CTTTT) are provided in SEQ ID NOs:427-435.
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M5 sequence set forth by SEQ ID NO: 16 (CAATG), a mutation in at least one nucleotide position of the wild type M6 set forth by SEQ ID NO: 17 (GGGTG) and at least one copy of the wild type M3 sequence set forth by SEQ ID NO: 21 (CTTTT) are provided in SEQ ID NOs:436-444.
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M4 sequence set forth by SEQ ID NO: 15 (CATTC), a mutation in at least one nucleotide of the wild type M5 sequence set forth by SEQ ID NO: 16 (CAATG), a mutation in at least one nucleotide position of the wild type M6 set forth by SEQ ID NO: 17 (GGGTG) and at least one copy of the wild type M3 sequence set forth by SEQ ID NO: 21 (CTTTT) are provided in SEQ ID NOs:445-451.
  • CATTC CATTC
  • CAATG a mutation in at least one nucleotide of the wild type M5 sequence set forth by SEQ ID NO: 16
  • GGGTG a mutation in at least one nucleotide position of the wild type M6 set forth by SEQ ID NO: 17
  • CTTTT at least one copy of the wild type M3 sequence set forth by SEQ ID NO: 21
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M4 sequence set forth by SEQ ID NO: 15 (CATTC), a mutation in at least one nucleotide position of the wild type M7 set forth by SEQ ID NO: 18 (ACTTT) and at least one copy of the wild type M3 sequence set forth by SEQ ID NO: 21 (CTTTT) are provided in SEQ ID NOs:452-458.
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M5 sequence set forth by SEQ ID NO: 16 (CAATG), a mutation in at least one nucleotide position of the wild type M7 set forth by SEQ ID NO: 18 (ACTTT) and at least one copy of the wild type M3 sequence set forth by SEQ ID NO: 21 (CTTTT) are provided in SEQ ID NOs:459-465.
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M4 sequence set forth by SEQ ID NO: 15 (CATTC), a mutation in at least one nucleotide of the wild type M5 sequence set forth by SEQ ID NO: 16 (CAATG), a mutation in at least one nucleotide position of the wild type M7 set forth by SEQ ID NO: 18 (ACTTT) and at least one copy of the wild type M3 sequence set forth by SEQ ID NO: 21 (CTTTT) are provided in SEQ ID NO:466.
  • CATTC CATTC
  • CAATG a mutation in at least one nucleotide position of the wild type M7 set forth by SEQ ID NO: 18
  • CTTTT at least one copy of the wild type M3 sequence set forth by SEQ ID NO: 21
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M4 sequence set forth by SEQ ID NO: 15 (CATTC), a mutation in at least one nucleotide position of the wild type M6 set forth by SEQ ID NO: 17 (GGGTG), a mutation in at least one nucleotide position of the wild type M7 set forth by SEQ ID NO: 18 (ACTTT) and at least one copy of the wild type M3 sequence set forth by SEQ ID NO: 21 (CTTTT) are provided in SEQ ID NOs:467-471.
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M5 sequence set forth by SEQ ID NO: 16 (CAATG), a mutation in at least one nucleotide position of the wild type M6 set forth by SEQ ID NO: 17 (GGGTG), a mutation in at least one nucleotide position of the wild type M7 set forth by SEQ ID NO: 18 (ACTTT) and at least one copy of the wild type M3 sequence set forth by SEQ ID NO: 21 (CTTTT) are provided in SEQ ID NOs:472-477.
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M4 sequence set forth by SEQ ID NO: 15 (CATTC), a mutation in at least one nucleotide of the wild type M5 sequence set forth by SEQ ID NO: 16 (CAATG), a mutation in at least one nucleotide position of the wild type M6 set forth by SEQ ID NO: 17 (GGGTG), a mutation in at least one nucleotide position of the wild type M7 set forth by SEQ ID NO: 18 (ACTTT) and at least one copy of the wild type M3 sequence set forth by SEQ ID NO: 21 (CTTTT) are provided in SEQ ID NOs:478-483.
  • CATTC CATTC
  • CAATG a mutation in at least one nucleotide position of the wild type M6 set forth by SEQ ID NO: 17
  • ACTTT a mutation in at least one nucleotide position of the wild type M7 set forth by S
  • the isolated polynucleotide further comprises at least one copy of the wild type M8 sequence set forth by SEQ ID NO: 19 (GCTTC) and at least one copy of the wild type M3 sequence set forth by SEQ ID NO: 21 (CTTTT) with additional wild type or mutated sequences derived from element X (SEQ ID NO:6).
  • Non-limiting examples of isolated polynucleotides which includes a mutation in at least one nucleotide of the wild type M4 sequence set forth by SEQ ID NO: 15 (CATTC), at least one copy of the wild type M8 sequence set forth by SEQ ID NO: 19 (GCTTC) and at least one copy of the wild type M3 sequence set forth by SEQ ID NO: 21 (CTTTT) are provided in SEQ ID NOs:484-495.
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M5 sequence set forth by SEQ ID NO: 16 (CAATG), at least one copy of the wild type M8 sequence set forth by SEQ ID NO: 19 (GCTTC) and at least one copy of the wild type M3 sequence set forth by SEQ ID NO: 21 (CTTTT) are provided in SEQ ID NOs:496-507.
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M4 sequence set forth by SEQ ID NO: 15 (CATTC), a mutation in at least one nucleotide of the wild type M5 sequence set forth by SEQ ID NO: 16 (CAATG), at least one copy of the wild type M8 sequence set forth by SEQ ID NO: 19 (GCTTC) and at least one copy of the wild type M3 sequence set forth by SEQ ID NO: 21 (CTTTT) are provided in SEQ ID NOs:508-515.
  • CATTC CATTC
  • CAATG a mutation in at least one nucleotide of the wild type M5 sequence set forth by SEQ ID NO: 16
  • GCTTC at least one copy of the wild type M8 sequence set forth by SEQ ID NO: 19
  • CTTTT at least one copy of the wild type M3 sequence set forth by SEQ ID NO: 21
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M4 sequence set forth by SEQ ID NO: 15 (CATTC), at least one copy of the wild type M6 set forth by SEQ ID NO: 17 (GGGTG), at least one copy of the wild type M8 sequence set forth by SEQ ID NO: 19 (GCTTC) and at least one copy of the wild type M3 sequence set forth by SEQ ID NO: 21 (CTTTT) are provided in SEQ ID NOs:516-519.
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M5 sequence set forth by SEQ ID NO: 16 (CAATG), at least one copy of the wild type M6 set forth by SEQ ID NO: 17 (GGGTG), at least one copy of the wild type M8 sequence set forth by SEQ ID NO: 19 (GCTTC) and at least one copy of the wild type M3 sequence set forth by SEQ ID NO: 21 (CTTTT) are provided in SEQ ID NOs:520-523.
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M4 sequence set forth by SEQ ID NO: 15 (CATTC), a mutation in at least one nucleotide of the wild type M5 sequence set forth by SEQ ID NO: 16 (CAATG), at least one copy of the wild type M6 set forth by SEQ ID NO: 17 (GGGTG), at least one copy of the wild type M8 sequence set forth by SEQ ID NO: 19 (GCTTC) and at least one copy of the wild type M3 sequence set forth by SEQ ID NO: 21 (CTTTT) are provided in SEQ ID NOs:524-525.
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M4 sequence set forth by SEQ ID NO: 15 (CATTC), at least one copy of the wild type M7 sequence set forth by SEQ ID NO: 18 (ACTTT), at least one copy of the wild type M8 sequence set forth by SEQ ID NO: 19 (GCTTC) and at least one copy of the wild type M3 sequence set forth by SEQ ID NO: 21 (CTTTT) are provided in SEQ ID NOs:526-529.
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M5 sequence set forth by SEQ ID NO: 16 (CAATG), at least one copy of the wild type M7 sequence set forth by SEQ ID NO: 18 (ACTTT), at least one copy of the wild type M8 sequence set forth by SEQ ID NO: 19 (GCTTC) and at least one copy of the wild type M3 sequence set forth by SEQ ID NO: 21 (CTTTT) are provided in SEQ ID NOs:530-533.
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M4 sequence set forth by SEQ ID NO: 15 (CATTC), a mutation in at least one nucleotide of the wild type M5 sequence set forth by SEQ ID NO: 16 (CAATG), at least one copy of the wild type M7 sequence set forth by SEQ ID NO: 18 (ACTTT), at least one copy of the wild type M8 sequence set forth by SEQ ID NO: 19 (GCTTC) and at least one copy of the wild type M3 sequence set forth by SEQ ID NO: 21 (CTTTT) are provided in SEQ ID NOs:534-535.
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M4 sequence set forth by SEQ ID NO: 15 (CATTC), at least one copy of the wild type M6 set forth by SEQ ID NO: 17 (GGGTG), at least one copy of the wild type M7 sequence set forth by SEQ ID NO: 18 (ACTTT), at least one copy of the wild type M8 sequence set forth by SEQ ID NO: 19 (GCTTC) and at least one copy of the wild type M3 sequence set forth by SEQ NO: 21 (CTTTT) are provided in SEQ ID NOs:536-537.
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M5 sequence set forth by SEQ ID NO: 16 (CAATG), at least one copy of the wild type M6 set forth by SEQ ID NO: 17 (GGGTG), at least one copy of the wild type M7 sequence set forth by SEQ ID NO: 18 (ACTTT) at least one copy of the wild type M8 sequence set forth by SEQ ID NO: 19 (GCTTC) and at least one copy of the wild type M3 sequence set forth by SEQ ID NO: 21 (CTTTT) are provided in SEQ ID NOs:538-539.
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M4 sequence set forth by SEQ ID NO: 15 (CATTC), a mutation in at least one nucleotide of the wild type M5 sequence set forth by SEQ ID NO: 16 (CAATG), at least one copy of the wild type M6 set forth by SEQ ID NO: 17 (GGGTG); at least one copy of the wild type M7 sequence set forth by SEQ ID NO: 18 (ACTTT), at least one copy of the wild type M8 sequence set forth by SEQ ID NO: 19 (GCTTC) and at least one copy of the wild type M3 sequence set forth by SEQ ID NO: 21 (CTTTT) are provided in SEQ ID NO:540.
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M4 sequence set forth by SEQ ID NO: 15 (CATTC), a mutation in at least one nucleotide position of the wild type M6 set forth by SEQ ID NO: 17 (GGGTG), at least one copy of the wild type M8 sequence set forth by SEQ ID NO: 19 (GCTTC) and at least one copy of the wild type M3 sequence set forth by SEQ ID NO: 21 (CTTTT) are provided in SEQ ID NOs:541-547.
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M5 sequence set forth by SEQ ID NO: 16 (CAATG), a mutation in at least one nucleotide position of the wild type M6 set forth by SEQ ID NO: 17 (GGGTG), at least one copy of the wild type M8 sequence set forth by SEQ ID NO: 19 (GCTTC) and at least one copy of the wild type M3 sequence set forth by SEQ ID NO: 21 (CTTTT) are provided in SEQ ID NOs:548-554.
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M4 sequence set forth by SEQ ID NO: 15 (CATTC), a mutation in at least one nucleotide of the wild type M5 sequence set forth by SEQ ID NO: 16 (CAATG), a mutation in at least one nucleotide position of the wild type M6 set forth by SEQ ID NO: 17 (GGGTG), at least one copy of the wild type M8 to sequence set forth by SEQ ID NO: 19 (GCTTC) and at least one copy of the wild type M3 sequence set forth by SEQ ID NO: 21 (CTTTT) are provided in SEQ ID NOs:555-559.
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M4 sequence set forth by SEQ ID NO: 15 (CATTC), a mutation in at least one nucleotide position of the wild type M7 set forth by SEQ ID NO: 18 (ACTTT), at least one copy of the wild type M8 sequence set forth by SEQ ID NO: 19 (GCTTC) and at least one copy of the wild type M3 sequence set forth by SEQ ID NO: 21 (CTTTT) are provided in SEQ ID NOs:560-566.
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M5 sequence set forth by SEQ ID NO: 16 (CAATG), a mutation in at least one nucleotide position of the wild type M7 set forth by SEQ ID NO: 18 (ACTTT), at least one copy of the wild type M8 sequence set forth by SEQ ID NO: 19 (GCTTC) and at least one copy of the wild type M3 sequence set forth by SEQ ID NO: 21 (CTTTT) are provided in SEQ ID NOs:567-573.
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M4 sequence set forth by SEQ ID NO: 15 (CATTC), a mutation in at least one nucleotide of the wild type M5 sequence set forth by SEQ ID NO: 16 (CAATG), a mutation in at least one nucleotide position of the wild type M7 set forth by SEQ ID NO: 18 (ACTTT), at least one copy of the wild type M8 sequence set forth by SEQ ID NO: 19 (GCTTC) and at least one copy of the wild type M3 sequence set forth by SEQ ID NO: 21 (CTTTT) are provided in SEQ ID NOs:574-578.
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M4 sequence set forth by SEQ ID NO: 15 (CATTC), a mutation in at least one nucleotide position of the wild type M6 set forth by SEQ ID NO: 17 (GGGTG), a mutation in at least one nucleotide position of the wild type M7 set forth by SEQ ID NO: 18 (ACTTT), at least one copy of the wild type M8 sequence set forth by SEQ ID NO: 19 (GCTTC) and at least one copy of the wild type M3 sequence set forth by SEQ ID NO: 21 (CTTTT) are provided in SEQ ID NOs:579-583.
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M5 sequence set forth by SEQ ID NO: 16 (CAATG), a mutation in at least one nucleotide position of the wild type M6 set forth by SEQ ID NO: 17 (GGGTG), a mutation in at least one nucleotide position of the wild type M7 set forth by SEQ ID NO: 18 (ACTTT), at least one copy of the wild type M8 sequence set forth by SEQ ID NO: 19 (GCTTC) and at least one copy of the wild type M3 sequence set forth by SEQ ID NO: 21 (CTTTT) are provided in SEQ ID NOs:584-588.
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of the wild type M4 sequence set forth by SEQ ID NO: 15 (CATTC), a mutation in at least one nucleotide of the wild type M5 sequence set forth by SEQ ID NO: 16 (CAATG), a mutation in at least one nucleotide position of the wild type M6 set forth by SEQ ID NO: 17 (GGGTG), a mutation in at least one nucleotide position of the wild type M7 set forth by SEQ ID NO: 18 (ACTTT), at least one copy of the wild type M8 sequence set forth by SEQ ID NO: 19 (GCTTC) and at least one copy of the wild type M3 sequence set forth by SEQ ID NO: 21 (CTTTT) are provided in SEQ ID NOs:589-592.
  • the isolated polynucleotide comprises at least one copy of wild type M3 sequence (SEQ ID NO: 21) and at least one copy of wild type M8 sequence (SEQ ID NO: 19), with at least one mutation in wild type M6 (SEQ ID NO: 17) and/or in wild type M7 (SEQ ID NO:50).
  • Non-limiting examples of isolated polynucleotides which include at least one copy of the wild type M8 sequence set forth by SEQ ID NO: 19 (GCTTC) and at least one copy of the wild type M3 sequence set forth by SEQ ID NO: 21 (CTTTT), with a mutation in at least one nucleotide of the wild type M6 sequence (SEQ ID NO: 17), and/or a mutation in at least one nucleotide of the wild type M7 (SEQ ID NO: 18) are provided in SEQ ID NOs:593-600.
  • an isolated polynucleotide which includes the wild type M8 sequence (SEQ ID NO: 19) and/or the wild type M3 (SEQ ID NO: 21) sequence in addition to tissue specific enhancers (e.g., wild type M4 and/or wild type M5), and/or induced enhancers (e.g., developmentally related- or stress related-enhancers) is expected to exert a more specific regulatory effect by suppressing expression in non-target cells or under non-induced conditions.
  • tissue specific enhancers e.g., wild type M4 and/or wild type M5
  • induced enhancers e.g., developmentally related- or stress related-enhancers
  • the isolated polynucleotide comprises at least one copy of the wild type M8 sequence set forth by SEQ ID NO: 19 (GCTTC) and an endothelial specific enhancer sequence.
  • the isolated polynucleotide comprises at least one copy of the wild type M8 sequence set forth by SEQ ID NO: 19 (GCTTC) and at least one copy of wild type M4 sequence set forth by SEQ ID NO: 15.
  • the isolated polynucleotide comprises at least one copy of the wild type M8 sequence set forth by SEQ ID NO: 19 (GCTTC) and at least one copy of wild type M5 sequence set forth by SEQ ID NO:16.
  • the isolated polynucleotide comprises at least one copy of the wild type M8 sequence set forth by SEQ ID NO: 19 (GCTTC), at least one copy of wild type M4 sequence set forth by SEQ ID NO: 15 and at least one copy of wild type M5 sequence set forth by SEQ ID NO:16.
  • the isolated polynucleotide comprises at least one copy of the wild type M3 sequence set forth by SEQ ID NO: 21 (CTTTT) and an endothelial specific enhancer sequence.
  • the isolated polynucleotide comprises at least one copy of the wild type M3 sequence set forth by SEQ ID NO: 21 (CTTTT) and at least one copy of wild type M4 sequence set forth by SEQ ID NO: 15.
  • the isolated polynucleotide comprises at least one copy of the wild type M3 sequence set forth by SEQ ID NO: 21 (CTTTT) and at least one copy of wild type M5 sequence set forth by SEQ ID NO:16.
  • the isolated polynucleotide comprises at least one copy of the wild type M3 sequence set forth by SEQ ID NO: 21 (CTTTT), at least one copy of wild type M4 sequence set forth by SEQ ID NO: 15 and at least one copy of wild type M5 sequence set forth by SEQ ID NO:16.
  • the isolated polynucleotide comprises at least one copy of the wild type M3 sequence set forth by SEQ ID NO: 21 (CTTTT), at least one copy of wild type M8 sequence set forth by SEQ ID NO: 19 (GCTTC) and an endothelial specific enhancer sequence.
  • the isolated polynucleotide comprises at least one copy of the wild type M3 sequence set forth by SEQ ID NO: 21 (CTTTT), at least one copy of wild type M8 sequence set forth by SEQ ID. NO: 19 (GCTTC) and at least one copy of wild type M4 sequence set forth by SEQ ID NO: 15.
  • the isolated polynucleotide comprises at least one copy of the wild type M3 sequence set forth by SEQ ID NO: 21 (CTTTT), at least one copy of wild type M8 sequence set forth by SEQ ID NO: 19 (GCTTC) and at least one copy of wild type M5 sequence set forth by SEQ ID NO: 16.
  • the isolated polynucleotide comprises at least one copy of the wild type M3 sequence set forth by SEQ ID NO: 21 (CTTTT), at least one copy of wild type M8 sequence set forth by SEQ ID NO: 19 (GCTTC), at least one copy of wild type M4 sequence set forth by SEQ ID NO: 15 and at least one copy of wild type M5 sequence set forth by SEQ ID NO: 16.
  • the isolated polynucleotide comprises at least one copy of the wild type M3 sequence set forth by SEQ ID NO: 21 (CTTTT), at least one copy of wild type M8 sequence set forth by SEQ ID NO: 19 (GCTTC) and at least one enhancer element such as wild type M6 (SEQ ID NO: 17) and/or wild type M7 sequence (SEQ ID NO:18).
  • the isolated polynucleotide includes at least one copy of wild type M8 with additional flanking sequences such as at least one copy of a wild type M8 sequence (SEQ ID NO:19), at least one copy of wild type M7 (SEQ ID NO: 18) and/or wild type M9 sequence (SEQ ID NO: 14, CTGGA); and/or the isolated polynucleotide includes at least one copy of wild type M8 and at least one mutation in M7, with or without M9 (SEQ ID NO: 22).
  • Such polynucleotides can be used as a non-specific repressor.
  • the isolated polynucleotide is for increasing expression of a heterologous polynucleotide operably linked thereto in cells/tissues.
  • the isolated polynucleotide comprises at least one copy of wild type M6 sequence set forth by SEQ ID NO: 17 (GGGTG) and/or at least one copy of wild type.
  • M7 sequence set forth by SEQ ID NO: 18 (ACTTT).
  • the isolated polynucleotide includes at least one copy of wild type M6 (SEQ ID NO: 17) and a mutation in at least one nucleotide of wild type M8 (SEQ ID NO: 19).
  • Non-limiting examples of isolated polynucleotide which include at least one copy of wild type M6 (SEQ ID NO: 17) and a mutation in at least one nucleotide of the wild type M8 (SEQ ID NO: 19) are provided in SEQ ID NOs:23-26.
  • the isolated polynucleotide includes at least one copy of wild type M7 (SEQ ID NO: 18) and a mutation in at least one nucleotide of wild type M8 (SEQ ID NO: 19).
  • Non-limiting examples of isolated polynucleotide which include at least one copy of wild type M7 (SEQ ID NO: 18) and a mutation in at least one nucleotide of the wild type M8 (SEQ ID NO: 19) are provided in SEQ ID NOs:27-28.
  • the isolated polynucleotide includes at least one copy of wild type M6 (SEQ ID NO: 17), at least one copy of wild type M7 (SEQ ID NO: 18) and a mutation in at least one nucleotide of wild type M8 (SEQ ID NO: 19).
  • the isolated polynucleotide includes at least one copy of wild type M1 (SEQ ID NO: 20) and a mutation in at least one nucleotide of wild type M8 (SEQ ID NO: 19).
  • Non-limiting examples of isolated polynucleotide which include at least one copy of wild type M1 (SEQ ID NO: 20) and a mutation in at least one nucleotide of the wild type M8 (SEQ ID NO: 19) are provided in SEQ ID NOs:43-54 and 601-632.
  • the isolated polynucleotide includes at least one copy of wild type M1 (SEQ ID NO: 20), at least one copy of wild type M6 (SEQ ID NO: 17) and/or at least one copy of wild type M7 (SEQ ID NO: 18) and a mutation in at least one nucleotide of wild type M8 (SEQ ID NO: 19).
  • Non-limiting examples of isolated polynucleotides which include a mutation in at least one nucleotide of wild type M8 (SEQ ID NO: 19) and at least one copy of wild type M1 (SEQ ID NO: 20), wild type M6 (SEQ ID NO: 17) and/or wild type M7 (SEQ ID NO: 18) are provided in SEQ ID NOs:29-42.
  • regulatory isolated polynucleotides which can be used according to some embodiments of the invention are provided (SEQ ID NOs: 633-644) in the Examples section which follows.
  • an isolated polynucleotide comprising a nucleic acid sequence which comprises a first polynucleotide comprising the pre-proendothelin (PPE-1) promoter set forth by SEQ ID NO:13 and a second polynucleotide comprising at least one copy of a nucleic acid sequence selected from the group consisting of:
  • the second polynucleotide is not SEQ ID NO:6 (element X), and wherein the isolated polynucleotide is not SEQ ID NO:12 (PPE-1-3X).
  • each of the wild type M4, M5, M8, M6, M7 and/or M1 sequences is placed in a head to tail (5′ ⁇ 3′) orientation with respect to the PPE-1 promoter set forth by SEQ ID NO:13.
  • each of the wild type M4, M5, M8, M6, M7 and/or M1 sequences is placed in a tail to head (3′ ⁇ 5′) orientation with respect to the PPE-1 promoter set forth by SEQ ID NO:13.
  • the wild type M4, M5, M8, M6, M7 and/or M1 sequences are placed in various orientations (head to tail or tail to head) and/or sequential order with respect the other wild type M4, M5, M8, M6, M7 and/or M1 sequences, and/or with respect to the orientation of SEQ ID NO:13.
  • Construction of such viral vectors may be effected using known molecular biology techniques such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992), in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989), Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich. (1995), Vega et al., Gene Targeting, CRC Press, Ann Arbor Mich. (1995), Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston Mass. (1988) and Gilboa et at. [Biotechniques 4 (6): 504-512, 1986].
  • the viral vector of this aspect of the present invention may be administered per se or as part of a pharmaceutical composition which also includes a physiologically acceptable carrier.
  • a pharmaceutical composition which also includes a physiologically acceptable carrier.
  • the purpose of a pharmaceutical composition is to facilitate administration of the active ingredient to an organism.
  • a “pharmaceutical composition” refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients.
  • the purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.
  • active ingredient refers to the viral vector of the present invention accountable for the biological effect.
  • physiologically acceptable carrier and “pharmaceutically acceptable carrier” which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound.
  • An adjuvant is included under these phrases.
  • excipient refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient.
  • excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
  • Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intracardiac, e.g., into the right or left ventricular cavity, into the common coronary artery, intravenous, inrtaperitoneal, intranasal, or intraocular injections. Injection of the viral vectors into a spinal fluid can also be used as a mode of administration.
  • sCAR-MTf melanotransferrin
  • Other approaches for enhancing the delivery of the virus to the CNS include pharmacological strategies designed to increase the lipid solubility of an agent (e.g., conjugation of water-soluble agents to lipid or cholesterol carriers); and the transitory disruption of the integrity of the BBB by hyperosmotic disruption (resulting from the infusion of a mannitol solution into the carotid artery or the use of a biologically active agent such as an angiotensin peptide).
  • an agent e.g., conjugation of water-soluble agents to lipid or cholesterol carriers
  • hyperosmotic disruption resulting from the infusion of a mannitol solution into the carotid artery or the use of a biologically active agent such as an angiotensin peptide.
  • the present invention also contemplates engineering of the viral vectors in order to avoid, suppress or manipulate the immune response, ideally resulting in sustained expression and immune tolerance to the transgene product—such methods are described for example in Nayak et al., Gene Therapy (12 Nov. 2009), incorporated herein by reference.
  • compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
  • the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.
  • physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • compositions may take the form of tablets or lozenges formulated in conventional manner.
  • the active ingredients for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
  • compositions described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative.
  • the compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.
  • a suitable vehicle e.g., sterile, pyrogen-free water based solution
  • compositions of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.
  • compositions suitable for use in context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients (i.e. viral particles) effective to prevent, alleviate or ameliorate symptoms of a disorder (e.g., glioblastoma) or prolong the survival of the subject being treated.
  • active ingredients i.e. viral particles
  • a disorder e.g., glioblastoma
  • the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays.
  • a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.
  • Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals.
  • the data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human.
  • the dosage may vary depending upon the dosage form employed and the route of administration utilized.
  • the exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch. 1 p. 1).
  • Dosage amount and interval may be adjusted individually to provide brain levels of the active ingredient are sufficient to induce or suppress the biological effect (minimal effective concentration, MEC).
  • MEC minimum effective concentration
  • the MEC will vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.
  • dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.
  • compositions to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.
  • unit dose refers to a physically discrete unit containing a predetermined quantity of an active material calculated to individually or collectively produce a desired effect such as an anti-cancer effect.
  • a single unit dose or a plurality of unit doses can be used to provide the desired effect, such as an anti-cancer therapeutic effect.
  • about 10 3 to about 10 16 virus particles are administered to the subject.
  • about 10 5 to about 10 13 virus particles are administered to the subject.
  • about 10 7 to about 10 12 virus particles are administered to the subject.
  • about 1 ⁇ 10 12 to about 5 ⁇ 10 12 virus particles are administered to the subject.
  • about 1 ⁇ 10 13 to about 5 ⁇ 10 13 virus particles are administered to the subject.
  • the subject is administered intravenously with 1 ⁇ 10 12 -1 ⁇ 10 13 viral particles of SEQ ID NO: 9. or SEQ ID NO: 10.
  • the subject is administered intravenously with at least two doses of 1 ⁇ 10 12 -1 ⁇ 10 13 viral particles of SEQ ID NO: 9. or SEQ ID NO: 10.
  • the subject is administered intravenously with at least three or more doses of 1 ⁇ 10 12 -1 ⁇ 10 13 viral particles of SEQ ID NO: 9. or SEQ ID NO: 10.
  • the at least two doses are administered at least about 1 day, at least about 3 days, at least about 5 days, at least about 7 days, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 2 months, at least about 6 months, at least about 9 months, at least about 1 year, at least about 1.25 years, at least about 1.5 years, at least about 1.75 years, at least about 2 years, at least about 2.5 years, at least about 3 years or more apart.
  • compositions of the present invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient.
  • the pack may, for example, comprise metal or plastic foil, such as a blister pack.
  • the pack or dispenser device may be accompanied by instructions for administration.
  • the pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert.
  • Compositions comprising a preparation of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as is further detailed above.
  • the vectors of the present invention may be administered with additional ingredients which may improve the uptake of the nucleic acid construct by the cells, expression of the chimeric polypeptide by the nucleic acid construct in the cells, or the activity of the expressed chimeric polypeptide.
  • adenoviral vectors into EC cells can be enhanced by treating the vectors with engineered antibodies or small peptides.
  • Such “adenobody” treatment was shown effective in directing adenovirus constructs to EGF receptors on cells (Watkins et at 1997, Gene Therapy 4:1004-1012).
  • Nicklin et at have shown that a small peptide, isolated via phage display, increased specificity and efficiency of vectors in endothelial cells and decreased the expression in liver cells in culture (Nicklin et at 2000, Circulation 102:231-237).
  • an FGF retargeted adenoviral vector reduced the toxicity of tk in mice (Printz et al 2000, Human Gene Therapy 11:191-204).
  • Low dose radiation has been shown to cause breaks in DNA strands primarily in the G2/M phase, cell membrane damage enhancing the bystander effect, and thus may potentiate other cytotoxic and anti-neoplastic therapies, when administered in combination.
  • Vascular endothelial cells may be particularly suitable to such combination, or adjunct, therapies, since it has been demonstrated that low dose radiation specifically targets the apoptotic system of the microvascular endothelial cells (Kolesnick et al., Oncogene 2003; 22:5897-906).
  • Angiostatin has been shown to potentiate the therapeutic effects of low dose radiation (Gorski et al. Can Res 1998; 58:5686-89).
  • the viral vectors and the pharmaceutical compositions comprising same of the present invention can be used to treat malignant gliomas alone or in combination with one or more other established or experimental therapeutic regimen for such disorders.
  • Therapeutic regimen for treatment of malignant gliomas suitable for combination with the viral vectors of the present invention include, but are not limited to chemotherapy, radiotherapy, phototherapy and photodynamic therapy, surgery, nutritional therapy, ablative therapy, combined radiotherapy and chemotherapy, brachiotherapy, proton beam therapy, immunotherapy, cellular therapy and photon beam radiosurgical therapy.
  • Anti-cancer drugs that can be co-administered with the compounds of the invention include, but are not limited to Acivicin; Aclarubicin; Acodazole Hydrochloride; Acronine; Adriamycin; Adozelesin; Aldesleukin; Altretamine; Ambomycin; Ametantrone Acetate; Aminoglutethimide; Amsacrine; Anastrozole; Anthramycin; Asparaginase; Asperlin; Azacitidine; Azetepa; Azotomycin; Batimastat; Benzodepa; Bicalutamide; Bisantrene Hydrochloride; Bisnafide Dimesylate; Bizelesin; Bleomycin Sulfate; Brequinar Sodium; Bropirimine; Busulfan; Cactinomycin; Calusterone; Caracemide; Carbetimer; Carboplatin; Carmustine; Carubicin Hydrochloride; Carzelesin; Cedefin
  • Additional antineoplastic agents include those disclosed in Chapter 52, Antineoplastic Agents (Paul Calabresi and Bruce A. Chabner), and the introduction thereto, 1202-1263, of Goodman and Gilman's “The Pharmacological Basis of Therapeutics”, Eighth Edition, 1990, McGraw-Hill, Inc. (Health Professions Division).
  • the viral vectors of the present invention may also be administered with an agent that enhances expression of transgenes in adenoviral-mediated transient expression.
  • an agent that enhances expression of transgenes in adenoviral-mediated transient expression For example International Application WO/2008/132729 teaches administration of a corticosteroid (e.g. dexamethasone and/or N-Acetyl Cysteine (NAC) prior to AdPPE-1 (3x)-Fas-c chimera construct administration.
  • a corticosteroid e.g. dexamethasone and/or N-Acetyl Cysteine (NAC) prior to AdPPE-1 (3x)-Fas-c chimera construct administration.
  • the viral vectors of the present invention may also be administered with an agent that brings about transient immunosuppression, such as for example deoxyspergualin (DSG) or cyclophosphamide (see for example Smith et al., Gene Ther. 1996 June; 3(6):496-502) in order to allow for repetitive dosing.
  • an agent that brings about transient immunosuppression such as for example deoxyspergualin (DSG) or cyclophosphamide (see for example Smith et al., Gene Ther. 1996 June; 3(6):496-502) in order to allow for repetitive dosing.
  • DSG deoxyspergualin
  • cyclophosphamide see for example Smith et al., Gene Ther. 1996 June; 3(6):496-502
  • chemotherapeutic agent is intended to include all such new technologies a priori.
  • compositions, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
  • a compound or “at least one compound” may include a plurality of compounds, including mixtures thereof.
  • range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
  • a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range.
  • the phrases “ranging/ranges between” a first indicate number and a second indicate number and “ranging/ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.
  • method refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
  • treating includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.
  • the vector was constructed using a backbone containing most of the genome of adenovirus type 5, as well as partial homology to an adaptor plasmid, which enables recombination.
  • the E1 early transcriptional unit was deleted from the backbone plasmid, and further modified by deleting the pWE25 and the Amp resistance selection marker site.
  • the adaptor plasmid containing sequences of the Ad5, CMV promoter, MCS, and SV40 polyA was modified to delete deleting the CMV promoter, and the PPE-1 promoter and Fas-c fragment were inserted by restriction digestion.
  • the modified PPE-1 promoter (PPE-1-3X, SEQ ID NO: 12) and the Fas-chimera transgene (Fas-c, SEQ ID NO: 4) were utilized for construction of the adenoviral vector.
  • the PPE-1-(3X)-Fas-c element (2115 bp) was constructed from the PPE-1-(3X)-luc element. This element contains the 1.4 kb of the murine preproendothelin PPE-1-(3X) promoter, the Luciferase gene, the SV40 polyA site and the first intron of the murine ET-1 gene, originated from the pEL8 plasmid (8848 bp) used by Harats et al (Harats D.
  • the PPE-3-Luc cassette was extracted from the pEL8 plasmid using the BamHI restriction enzyme.
  • the Luciferase gene was substituted by the Fas-c gene [composed of the extra cellular and intra membranal domains of the human TNF-R1 (Tumor Necrosis Factor Receptor 1, SEQ ID NO: 2) and of the Fas (p55) intracellular domain (SEQ ID NO: 3) (Boldin et al, JBC, 1995)] to obtain the PPE-1-3x-Fas-c cassette.
  • PPE-1(3x)-Fas-c Plasmid The cassette was further introduced into the backbone plasmid by restriction digestion, resulting with the PPE-1(3x)-Fas-c plasmid.
  • Adaptor-PPE-1(3x)-Fas-c Plasmid The PPE-1-3x-Fas-c element was extracted from the first generation construct PPE-1-3x-Fas-c plasmid, and was amplified with designated PCR primers introducing SnaB1 and EcoR1 restriction sites at the 5′-and-3′-end respectively. These sites were used to clone the PPE-Fas-c fragment into the adaptor plasmid digested with SnaB1 and EcoR1, resulting in the adaptor-PPE-1-3x-Fas-c used for transfection of the host cells (for example, PER.C6 cells).
  • a small burr hole was made in the cranium using a mounted Micromotor drill (Stoelting) at the identified position (1 mm forward and 4 mm lateral of the bregma).
  • 1 ⁇ 10 6 cells in a volume of 5 ul were injected into the caudate over 5 minutes using a Quintessential Stereotaxic Injector (Stoelting) containing a 10 ul Hamilton syringe mounted to the stereotaxic device to assure appropriate placement.
  • Animals were imaged following isofluorane sedation using an IVIS chemiluminescence system. The fluorescence/bioluminescence of these tumors are typically detectable within 7-10 days due to rapid growth and high expression of the marker.
  • An alternative imaging modality i.e.
  • MRI Magnetic resonance Imaging
  • Animals received chemical anesthesia for MRI imaging. Once tumor establishment and growth was detected (variable depending on the rate of growth for the respective line), rats were treated with VB-111. The total dose was 10 11 vp in a volume of 100 ul. Control groups received vehicle only. Animals were monitored for tumor growth or response through non-invasive imaging of fluorescence/luminescence—see FIG. 1 for a typical treatment and monitoring regimen.
  • TGI tumor growth inhibition
  • TTD tumor growth delay
  • Magnetic resonance imaging has shown to be capable of demonstrating early changes within the tumor vasculature without any invasive measures. It is possible to generate maps of blood volume and blood flow, vascular permeability, white matter tracks, and apparent diffusion coefficient. These parameters offer clinically relevant physiological information that could help to characterize, stage tumor growth, and evaluate treatment efficacy.
  • MRI was performed on a Bruker 7 Tesla scanner. Blood flow and blood volume was measured using dynamic contrast enhanced imaging technique following a bolus of gadopentetate-dimeglumine (GdDTPA). White matter tracks and apparent diffusion coefficient was measured using diffusion tensor imaging. Vascular permeability was measured using T1-weighted MRI obtained prior to and following contrast (Gd-DTPA) injection.
  • EPI gradient echo planar imaging
  • repetition time TR 2 s
  • echo time TE 40 ms
  • b value 0 s/mm2
  • 6 diffusion direction of b 1100 s/mm2.
  • the number of slices analyzed adequately covered the entire tumor region and roughly cover the entire cerebrum.
  • Ktrans can be influenced by flow, or by permeability, or both.
  • flow limitations are not usually a concern, but the blood-brain barrier severely limits permeability unless it is disrupted by disease.
  • Ktrans does not fully correspond to permeability, but it is related rather to the permeability*surface area product of the capillary bed (in nonflow-limited situations).
  • mice were sacrificed by cardiac puncture, followed by intracardiac saline and formalin irrigation. Necropsy was performed, and brains underwent standard H&E processing. The number of vessels per medium power field were counted.
  • animal death began at approximately day 32.
  • the median survival for the control group was 39.25 (+/ ⁇ 3.8) days and for the treatment group was 45.8 days.
  • Luciferase activity was followed by ip injection of luciferin and optical imaging on a Xenogen system. The region of interest was generated automatically without manipulation and total photons recorded. As illustrated in FIG. 3 , a clear separation in activity was observed at day 33 with a mean (SD) in the control group of 9.7 (2.9) ⁇ 10 6 versus 5.3 (6.2) ⁇ 10 5 in the treated group.
  • SD mean
  • mean of the maximum diameters of tumors in the VB111 treated group was smaller than those for controls.
  • VB-111 will be administered as a single intravenous infusion of 1 ⁇ 10 12 or 3 ⁇ 10 12 Dose.
  • Study consists of 2 cohorts.
  • Cohort 1a 3-6 subjects, safety (1 ⁇ 10 12 VPs);
  • Cohort 1b 3-6 subjects, safety (3 ⁇ 10 12 VPs);
  • Cohort 1a & Cohort 1b Study subjects will be enrolled sequentially. The first subject of each cohort will be treated and observed for 14 days; if no dose-limiting toxicities (DLT) are observed, then another two subjects will be recruited to that cohort. All six subjects of cohort 1 need to be observed for a minimum of 14 days and show no DLT for the start of the next cohort. If a DLT is observed in one patient in a specific dosing cohort, three additional subjects will be accrued for the same dosing cohort, and safety will be reassessed. If DLT is confirmed, i.e. two out of six subjects experience a DLT, then the study will be discontinued. All subjects in cohorts 1a and 1b must be observed for a minimum of 28 days prior to commencing cohort 2.
  • DLT dose-limiting toxicities
  • the study will be conducted according to the Simon's 2 step method.
  • a total of 29 subjects are anticipated to enroll at the 3 ⁇ 10 12 VP dose level (3-6 from cohort 2 and 23-26 in cohort 3).
  • Step one will include the first 10 patients at this dose level.
  • a subject will be considered to have a response if s/he has either 6 months progression free survival or at least a partial tumor response according to Rano criteria.
  • An interim analysis will be performed after 10 patients from cohorts 2 and 3 have completed the study. If 2 or more responses occur in the step 1 subjects, step 2 will commence, enrolling an additional 19 subjects.
  • VB 111 (1 ⁇ 10 12 , 3 ⁇ 10 12 or 1 ⁇ 10 13 VPs) administered within 3 weeks after the visit. Subjects will return to the clinic for follow up visits at days 4, 7, 14 and 28 and on monthly schedule on days 56, 84, 112, 140 and 168 if no disease progression has occurred prior to the visit.
  • subjects will be assessed for response using contrast and non-contrast brain magnetic resonance imaging (MRI) with assessment based on the RANO criteria.
  • MRI contrast and non-contrast brain magnetic resonance imaging
  • the post study follow up period will include telephone contacts every two months after day 168, early termination, or disease progression (whichever occurs earlier) to follow up on survival. follow up will continue until patient expires.
  • the study duration is 7 months (6 months post dose), thereafter the subjects will be followed by telephone for survival data every two months.
  • Surveillance MRIs will be performed every 2 months until 1 year, and then every 3 months until 2 years post dosing (or until progression).
  • Subjects must have histologically confirmed diagnosis of primary malignant glioma (glioblastoma multiforme, gliosarcoma or anaplastic astrocytoma, or anaplastic oligodendroglioma). Subjects with recurrent disease whose diagnostic pathology confirmed malignant glioma (glioblastoma multiforme, gliosarcoma or anaplastic astrocytoma, or anaplastic oligodendroglioma) will not need re-biopsy.
  • TKIs such as sunitinib or sorafenib
  • Subjects must be treated with corticosteroids on day 0. Subjects will be on a stable dose for 1 week prior to entry, and is not anticipated to require increase in steroid dose throughout the study.
  • Prior anti-angiogenic therapy including VEGF-sequestering agents (e.g. bevicizumab, aflibercept) or VEGF inhibitors (e.g. cedirinib, pazopanib, sunitinib, sorafenib).
  • VEGF-sequestering agents e.g. bevicizumab, aflibercept
  • VEGF inhibitors e.g. cedirinib, pazopanib, sunitinib, sorafenib.
  • liver disease alcoholic, drug/toxin induced, genetic, or autoimmune.
  • Subjects may not have received any other investigational agent within 4 weeks before enrolment.
  • Uncontrolled intercurrent illness including, but not limited to ongoing or active infection, symptomatic congestive heart failure, unstable angina pectoris, cardiac arrhythmia, or psychiatric illness/social situations that would limit compliance with study requirements
  • VB-111 (SEQ ID NO: 9 or 10) is formulated as a sterile vector solution.
  • the solution is supplied frozen (below ⁇ 65° C.), in single use, plastic screw cap vials. Each vial contains 1.1 mL of vector solution at a viral titer of 1 ⁇ 10 12 VP/ml.
  • the vector solution should be thawed and maintained on ice during dilution and handling for a maximum of 3 hours.
  • the solution for injection Prior to infusion, the solution for injection should be brought to room temperature Maximum time for drug in saline is 1 hour at room temperature.
  • the vials should be opened in a biological safety cabinet and injected into 4 mL of normal saline for infusion for each 1 ml of drug. ie; for the 1 ⁇ 10 12 viral particle (VP) dose 1 ml of drug+4 ml of saline, for the 3 ⁇ 10 12 VP dose 3 ml of drug+12 ml of saline.
  • a single infusion of approximately 5 mL/15 ml of diluted VB-111 should be administered 1 mL/minute.
  • Adverse events will be recorded on an ongoing basis and up to 2 months following the administration of the test drug. Adverse events will be assessed for seriousness, relatedness to study drug, and severity (according to CTCAE 4.0). Vital signs will be recorded at screening, prior to dosing, 30, 60 minutes, 4 and 6 hours after dosing and at all patient visits. A physical examination will be conducted at screening, days 14, 28, 56, 84, 112, 140, 168 and at the end of the study. A 12 lead ECG will be obtained at screening, prior to dosing and on days 28 and 168 (or ET). Safety laboratory assessment (blood haematology and chemistry, urine analysis) will be conducted at screening, prior to dosing, and at all patient visits, starting from day 4 ⁇ 1 to 168 ⁇ 7.
  • Blood and urine samples will be collected prior to dosing, at the end of the infusion, days 4, 7, 14, 28 and 56, for evaluation of levels of virus DNA (in whole blood and urine) and its transgene (in whole blood).
  • Tumor response will be assessed at screening, prior to dosing, days 14, 28, 56, 112, 140 and 168, and then every 2 months for 1 year and every 3 months for 2 years post dosing, using contrast and non-contrast brain magnetic resonance imaging (MRI) with assessment based on the RANO criteria, until progression of disease (local and central independent radiology review).
  • MRI contrast and non-contrast brain magnetic resonance imaging
  • PFS will be censored at the time of initiation of alternative anticancer therapy, date of last radiologic assessment, or time of last contact.

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